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Extracellular Human Immunodeficiency Virus Type-1 Tat Protein Activates Phosphatidylinositol 3-Kinase in PC12 Neuronal Cells*

  • Daniela Milani
    Affiliations
    Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy and the

    Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02215
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  • Meri Mazzoni
    Affiliations
    Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy and the
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  • Paola Borgatti
    Affiliations
    Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy and the

    Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02215
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  • Giorgio Zauli
    Affiliations
    Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy and the
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  • Lewis Cantley
    Affiliations
    Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02215
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  • Silvano Capitani
    Correspondence
    To whom all correspondence should be addressed
    Affiliations
    Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy and the
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  • Author Footnotes
    * This research was supported by the AIDS Target Project of the Italian Ministry of Health and by National Institutes of Health Grant GM41890 (to L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 20, 1996DOI:https://doi.org/10.1074/jbc.271.38.22961
      We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum-starved PC12 cells. Moreover, the addition of Tat to PC12 cells induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 n wortmannin completely blocked the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.

      INTRODUCTION

      The regulatory HIV-1
      The abbreviations used are: HIV-1s
      human immunodeficiency virus type-1
      PI 3-kinase
      phosphatidylinositol 3-kinase
      anti-Tyr(P)
      anti-phosphotyrosine
      PBS
      phosphate-buffered saline
      WT
      wortmannin
      PMSF
      phenylmethylsulfonyl fluoride
      TBS
      Tris-buffered saline
      PI
      phosphatidylinositol
      HPLC
      high performance liquid chromatography
      PI 4-kinase
      phosphatidylinositol 4-kinase
      NGF
      nerve growth factor
      mAb
      monoclonal antibody.
      Tat protein is a small (86-104 amino acid) protein encoded by two exons. Tat can be divided in five distinct domains called N-terminal, cysteine-rich, core, basic, and C-terminal sequences. While the cysteine-rich region is responsible for the formation of intramolecular disulfide bonds, the basic region contains nuclear localization signals and the binding site for the transactivation response element RNA, located at the 5′ end of all viral mRNAs (
      • Jones K.A.
      • Peterlin M.B.
      ).
      The best characterized biological effect of Tat is the transactivation of HIV-1 genome, which takes place after specific interactions of Tat protein with the stem-loop transactivation response element sequence of the long terminal repeat viral RNA at the nuclear level. In addition to acting intracellularly, Tat protein shows the unique property, for a viral protein, to be actively released in culture by HIV-1-infected and tat-transfected cells (
      • Ensoli B.
      • Barillari G.
      • Zaki Salahuddin S.
      • Gallo R.C.
      • Wong-Staal F.
      ) and displays pleiotropic activities on the survival, growth, and function of various cell types by acting as a viral growth factor (
      • Ensoli B.
      • Barillari G.
      • Zaki Salahuddin S.
      • Gallo R.C.
      • Wong-Staal F.
      ,
      • Milani D.
      • Zauli G.
      • Neri L.M.
      • Marchisio M.
      • Previati M.
      • Capitani S.
      ,
      • Zauli G.
      • Gibellini D.
      • Milani D.
      • Mazzoni M.
      • Borgatti P.
      • La Placa M.
      • Capitani S.
      ,
      • Viscidi R.P.
      • Mayur K.
      • Lederman H.M.
      • Frankel A.D.
      ,
      • Li C.J.
      • Friedman D.J.
      • Wang C.
      • Metelev V.
      • Pardee A.B.
      ). In particular, we have previously demonstrated that picomolar concentrations of recombinant Tat (0.1-10 ng/ml) promote the survival of PC12 rat pheochromocytoma cells under serum-free culture conditions (
      • Milani D.
      • Zauli G.
      • Neri L.M.
      • Marchisio M.
      • Previati M.
      • Capitani S.
      ). Additionally, PC12 cells stably transfected with tat cDNA show an increased resistance to apoptotic death (
      • Zauli G.
      • Gibellini D.
      • Milani D.
      • Mazzoni M.
      • Borgatti P.
      • La Placa M.
      • Capitani S.
      ). However, the addition in culture of anti-Tat neutralizing antibodies to these tat-transfected cells completely blocked their increased resistance to apoptosis (
      • Zauli G.
      • La Placa M.
      • Vignoli M.
      • Re M.C.
      • Gibellini D.
      • Furlini G.
      • Milani D.
      • Marchisio M.
      • Mazzoni M.
      • Capitani S.
      ), indicating that Tat released extracellularly by tat-transfected cells is required to promote cell survival.
      Although experimental evidence for the existence of high affinity receptor for Tat is still lacking, our data suggested that recombinant Tat might act by inducing intracellular signal transduction. Since it has been recently shown that NGF protects PC12 cells from apoptosis by activating PI 3-kinase (
      • Yao R.
      • Cooper M.G.
      ), we here investigated whether extracellular Tat was also able to activate PI 3-kinase in PC12 cells.

      DISCUSSION

      In this report, we have demonstrated that extracellular HIV-1 Tat protein promotes the activation of PI 3-kinase in PC12 rat pheochromocytoma cells. PI 3-kinase phosphorylates the D-3 position of the inositol ring of phosphoinositols and produces D-3 phosphoinositides (phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate). Increasing experimental evidence indicates that D-3 phosphorylated inositides represent a class of second messenger molecules, which can activate specific protein kinase C isoforms and Akt-Ser/Thr kinase, interact with SH2 domains and induce cytoskeletal rearrangement (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kaziauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kapeller R.
      • Chakrabarti R.
      • Cantley L.C.
      • Fay F.
      • Corvera S.
      ). PI 3-kinase is often found in cellular complexes with ligand-activated growth factor receptors and oncogene protein-tyrosine kinases.
      Activation of the mammalian PI 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex has been implicated in a variety of cell functions, including mitogenesis, cell transformation, etc. (
      • Kapeller R.
      • Cantley L.C.
      ). Moreover, a role for PI 3-kinase in the promotion of neuronal cell survival has been recently demonstrated by Yao and Cooper (
      • Yao R.
      • Cooper M.G.
      ). These authors have shown that NGF counteracts the apoptotic cell death program induced by serum withdrawal in PC12 cells by activating PI 3-kinase. In this respect, it is particularly noteworthy that we have previously demonstrated that recombinant Tat is also able to protect PC12 cells from apoptosis induced by serum withdrawal (
      • Zauli G.
      • Gibellini D.
      • Milani D.
      • Mazzoni M.
      • Borgatti P.
      • La Placa M.
      • Capitani S.
      ).
      We have shown here that 1 ng/ml Tat potently stimulates PI 3-kinase, reaching values of activation (approximately 20-fold increase with respect to the basal level) significantly higher than those observed in PC12 cell cultures supplemented with 100 ng/ml NGF (8-fold increase with respect to the basal level). Therefore, PI 3-kinase may represent a common intracellular target for both NGF and Tat protein.
      It is also noteworthy that activation of PI 3-kinase was achieved with picomolar concentrations of recombinant Tat, which are likely to be physiologically present in vivo, since similar concentrations have been detected in the supernatant of HIV-1-infected cells (
      • Ensoli B.
      • Barillari G.
      • Zaki Salahuddin S.
      • Gallo R.C.
      • Wong-Staal F.
      ,
      • Zauli G.
      • La Placa M.
      • Vignoli M.
      • Re M.C.
      • Gibellini D.
      • Furlini G.
      • Milani D.
      • Marchisio M.
      • Mazzoni M.
      • Capitani S.
      ) as well as in the sera of some HIV-1-infected individuals (
      • Westendorp M.O.
      • Frank R.
      • Ochsenbauer C.
      • Stricker K.
      • Dhein J.
      • Walczak H.
      • Debatin K.-M.
      • Krammer P.H.
      ). On the other hand, 0.1-1 μg/ml Tat protein were less efficient in promoting PI 3-kinase activation. Consistently, these high concentrations of Tat were unable to promote cell survival and instead showed a toxic effect on both neuronal and lymphoid cell types (
      • Li C.J.
      • Friedman D.J.
      • Wang C.
      • Metelev V.
      • Pardee A.B.
      ,
      • Westendorp M.O.
      • Frank R.
      • Ochsenbauer C.
      • Stricker K.
      • Dhein J.
      • Walczak H.
      • Debatin K.-M.
      • Krammer P.H.
      ,
      • Magnuson D.S.
      • Knudsen B.E.
      • Geiger J.D.
      • Brownstone R.M.
      • Nath A.
      ). This cytotoxic activity was likely due to the up-regulation of cellular genes encoding for inhibitory cytokines (
      • Zauli G.
      • Davis B.R.
      • Re M.C.
      • Visani G.
      • Furlini G.
      • La Placa M.
      ,
      • Buonaguro L.
      • Barillari G.
      • Chang H.K.
      • Bohan C.A.
      • Kao V.
      • Morgan R.
      • Gallo R.C.
      • Ensoli B.
      ).
      Since extracellular Tat can be rapidly taken up by intact cells and concentrate into the nucleus (
      • Frankel A.D.
      • Pabo C.O.
      ), a possible explanation for many of the biological effects of Tat, including the prevention from apoptosis, is a direct gene transactivation (
      • Mann D.A.
      • Frankel A.D.
      ). However, several lines of evidence suggest that extracellular Tat may also specifically interact with surface receptors. Although the demonstration of a high affinity receptor for extracellular Tat protein is still lacking, various integrins (αvβ3, α5β1, αvβ5) have been proposed as putative receptors for extracellular Tat (
      • Brake D.A.
      • Debouck C.
      • Biesecher G.
      ,
      • Barillari G.
      • Gendelman R.
      • Gallo R.C.
      • Ensoli B.
      ,
      • Vogel B.E.
      • Lee S.J.
      • Hildebrand A.
      • Craig W.
      • Pierschbacher M.D.
      • Wong-Staal F.
      • Ruoslahti E.
      ). Moreover, Weeks et al. (
      • Weeks B.S.
      • Desai K.
      • Loewenstein P.M.
      • Klotman M.E.
      • Klotman P.
      • Green M.
      • Kleinman H.K.
      ) have described the existence of a 90-kDa surface receptor that was specifically immunoprecipitated by an anti-Tat mAb from the surface of PC12 cells. Several considerations favor the hypothesis that extracellular Tat activates PI 3-kinase acting through a surface receptor. (i) Various integrins have been shown to activate protein-tyrosine kinases, such as p125FAK (
      • Zachary I.
      • Rozengurt E.
      ). Consistently, we have shown that Tat induced an early (1 min) tyrosine phosphorylation of the p85 regulatory subunit of PI 3-kinase, which acts as an adaptor protein allowing the p110 catalytic subunit to interact with receptor and nonreceptor protein-tyrosine kinases and tyrosine-phosphorylated proteins. (ii) Maximal activation of PI 3-kinase was obtained with concentrations of Tat as low as 1 ng/ml, while higher concentrations were less efficient in activating PI 3-kinase. This renders less likely the possibility that Tat must be internalized in order to activate PI 3-kinase.
      In conclusion, this is the first report demonstrating that HIV-1 Tat protein, which is actively released by infected cells, is able to generate intracellular signals activating PI 3-kinase.

      Acknowledgments

      We thank Dr. Stephen Soltoff for helpful discussions.

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